Means and methods for active cellular immunotherapy of cancer by using tumor cells killed by high hydrostatic pressure and dendritic cells

ABSTRACT

Disclosed are pharmaceutical compositions for inducing an immune response against tumor cells comprising tumor cells which are made apoptotic by treatment with high hydrostatic pressure and dendritic cells, and methods for producing such compositions.

PRIORITY

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/504,387 filed Jul. 5, 2011, the entire contentsof which are incorporated by reference herein.

BACKGROUND OF THE PRESENT INVENTION

Diseases caused by different tumors are still major problems in medicineand human health. The combination of surgery, chemotherapy andradiotherapy greatly improved the prognosis of cancer patients. Despitethat this approach results frequently in a significant reduction oftumor mass, a small population of precursor tumor cells or cancer stemcells often survives and subsequently gives rise to a new population oftumor cells that leads to a relapse. Even if the main tumor is removedby surgical and/or other treatments minor amounts of circulating tumorcells may cause metastatic tumors in different areas of the body.Therefore, there exists a permanent need for alternative medicaments andmethods of treatment which may be used alone or preferably be combinedwith other methods of tumor treatment.

PRIOR ART

WO 2006/095330 describes methods for inhibiting growth of cellpopulations by thermally, mechanically and/or chemically damagingantigen-bearing cells and introducing said cells as aggregate withantigen-presenting cells into patients.

Frank et al. “Harnessing Naturally Occurring Tumor Immunity; A ClinicalVaccine Trial in Prostate Cancer”, PLOS ONE, vol. 5, no. 9, 1 January2010 (2010-01-01), page E12367, disclose a tumor vaccine comprisingautologous dendritic cells and apoptotic UV-irradiated LNCaP cells.

Minarik et al. “Phase I/II of Clinical Study of Prostate CancerImmunotherapy Using Dendritic Cell Vaccination Strategy—First Results”,European Urology Supplements, vol. 9, no. 6, 1 Sep. 2010, page 629,disclose the preliminary results of a phase I/II clinical study ofprostate cancer immunotherapy using dendritic cells pulsed withapoptotic LNCaP cells killed by UVA irradiation.

Weiss et al. “Ex vivo- and in vivo-induced dead tumor cells asmodulators of antitumor responses”, Annals of the New York Academy ofSciences, vol. 1209, no. 1, 1 Oct. 2010 (2010-10-01), pages 109-117,disclose high hydrostatic pressure treatment of tumor cells. The deadtumor cells are directly used as cancer vaccines in animal models.

Weiss et al. “High hydrostatic pressure treatment generates inactivatedmammalian tumor cells with immunogeneic features”, Journal ofImmunotoxicology, vol. 7, no. 3, 1 Sep. 2010, pages 194-204, disclosethat high hydrostatic pressure treatment induces apoptosis wherein tumorcells are inactive and immunogenic. Antibodies directed against tumorcells have been produced and identified in a mouse model.

US 2008/0286314 discloses cancer vaccines comprising antigen presentingcells loaded with heat-shocked cancer cells which are non-apoptoticwhich can be used for treating cancer patients.

SUMMARY OF THE PRESENT INVENTION

The present invention relates to pharmaceutical compositions which canbe used for the induction of anti-tumor immune response, in particularin tumor vaccination causing the body to produce an immunogenic reactionagainst tumor cells.

Tumor cells killed by standard modalities such as irradiation arenormally non-immunogenic. If used for the generation of cancerimmunotherapy products, irradiated tumor cells need to be administeredin combination with a potent adjuvant. When used for pulsing of antigenpresenting cells, such as dendritic cells, irradiated killed tumor cellsdo not provide an activating signal. Dendritic cells thus need to beactivated by another substance, such as pathogen derived molecules.

A novel process is disclosed that induces an immunogenic death of humantumor cells, in particular ovarian and prostate cancer cells and acutelymphoblastic leukemia cells of human origin. Tumor cells killed by highhydrostatic pressure (in the following also: HHP) provide a potentactivation stimulus to dendritic cells, in particular to immaturedendritic cells, even in the absence of additional stimuli. Tumor cellskilled by this method express high levels of immunogenic cell deathmarkers and dendritic cells loaded with those immunogenic tumor cellsinduce high numbers of tumor specific T lymphocytes without expandingundesirable regulatory T lymphocytes. The experimental data of thepresent invention show that the combination of tumor cells killed by theapplication of high hydrostatic pressure and dendritic cells results inthe phagocytosis and efficient presentation of tumor antigens and in theinduction of strong anti-tumor immune responses.

Tumor cells are not or only weakly immunogenic and they usually do nothave the capacity to induce a tumor specific immune response if used inthe absence of a powerful adjuvant. Recent studies have shown that tumorcells killed by some chemotherapeutics, such as bortezomib, oxaliplatinand anthracyclines, can induce a tumor-specific immune response. Thisimmunogenic cell death is characterized by molecular events shared forall described chemotherapeutics. Within hours after the initiation ofimmunogenic cell death, preapoptotic tumor cells translocatecalreticulin and heat shock proteins from the endoplasmic reticulum tothe cell surface together with other molecules that serve as ‘eat me’signals (phosphatidylserine).

At the same time, tumor cells undergoing immunogenic tumor cell deathdownregulate the expression of ‘don't eat me’ signals (such as surfaceCD47) to facilitate tumor-cell recognition and engulfment by dendriticcells. Additionally, following permeabilization of the plasma membrane,cells release the late apoptosis marker high mobility group box 1(HMGB1) into the extracellular milieu. HMGB1 can bind several patternrecognition receptors (PRRs), such as Toll-like receptor 2 (TLR2), TLR4and receptor for advanced glycosylation end products (RAGE). The releaseof this protein seems to be required for optimal presentation ofantigens from dying tumor cells, T-cell priming by dendritic cells andsubsequent T-cell-mediated elimination of the tumor.

Use of tumor cells killed in such a way that they become immunogenic isextremely important for the design of cancer immunotherapeuticstrategies. Administration of immunogenic tumor cells can induce a tumorspecific immune response that will then control the growth of tumorcells. This will slow down or even stabilize the progression of thedisease and improve the prognosis of cancer patients. It is also assumedthat the distribution of tumor cells circulating in the body and theformation of metastases can be at least substantially reduced.

PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

A novel method and pharmaceutical compositions are disclosed that inducean immunogenic cell death of human tumors, in particular ovarian andprostate cancer cells and acute lymphoblastic leukemia cells to a muchhigher extent than recently described chemotherapeutics. Tumor cellskilled by this method and captured by dendritic cells express highlevels of immunogenic cell death markers and induce high numbers oftumor specific T lymphocytes without inducing regulatory T cells thatcould inhibit anti-tumor immune response. It has been found that thedegree of the anti-tumor immune response obtained by the combination oftumor cells treated according to the present invention and dendriticcells is about 10-fold higher than the immune response induced byimmunogenic tumor cells alone.

The general principle of a preferred cancer immunotherapy protocol basedon the administration of mature dendritic cells (DCs) loaded with killedtumor cells is shown in FIG. 1. All steps of the generation of the finalpharmaceutical composition are performed under Good ManufacturingPractice conditions in GMP facility.

In a preferred embodiment the first step in the process of generation ofthe pharmaceutical composition for each patient is a leukapheresisperformed for the purpose of collecting large numbers of monocytes fromthe peripheral blood. In a preferred embodiment the leukaphereticproduct is then diluted in a suitable buffer, such as PBS+1 mM EDTA(Lonza, Vierviers, Belgium) and mononuclear cells are separated byPremium Ficoll Paque (GE Healthcare, Little Chalfont, UK) gradientcentrifugation. Collected mononuclear cells (PBMC) are then washed [e.g.in PBS+1 mM EDTA (Lonza)], resuspended in Cell Gro medium and plated intriple flasks (e.g. NUNC, Roskilde, Denmark) at 1×10⁶ cells per cm² ofsurface area. After two hours non-adherent cells are washed with PBS(Lonza). Adherent monocytes are cultured for 6 days in Cell Gro mediumwith 20 ng/ml of IL-4 (Gentaur) and 500 U/ml of GM-CSF (Gentaur), freshcytokines are added on day 3.

Immature DCs are harvested on day 6 and loaded with killed tumor cells(e.g. prostate cancer cell line, ovarian cancer cell line, acutelymphoblastic leukemia cell line). Freshly thawed, immature DCs (day3-6) are fed with tumor cells at a fixed DC: tumor cell ratio of 5:1 for4 h. The ratio of dendritic cells to treated tumor cells is preferablywithin a range between 1:1 up to 10:1, more preferred between about 4:1and 6:1.

According to the present invention dendritic cells which are in variousstages of differentiation, maturation and/or activation can be used. Thematuration stage of the dendritic cells can be influenced by maturationfactors.

Tumor cell-pulsed DCs are then preferably matured by 25 μg/ml of PolyI:C during overnight incubation and cryopreserved and stored in liquidnitrogen. Before administration, 1×10⁷ mature DCs pulsed with tumorcells are resuspended in 0.9% NaCl (Baxter) and injected subcutaneouslyin the inguinal and brachial area within 12 hours preferably from 30minutes up to 12 hours. Administration of this form of cancerimmunotherapy is preferably repeated in regular intervals of 2-6 weeksin order to continuously boost the immune response. It is assumed thatthe method disclosed herein prevents the reestablishment oftumor-induced immune tolerance. The therapeutic efficacy of this form ofimmunotherapy has been documented in patients with prostate cancer indistinct clinical stages, biochemical relapse of the prostate cancer andcastration resistant metastatic prostate cancer, presumably also inmetastatic hormone-sensitive stage.

The above-described preferred embodiment is, however, in no waylimiting. In the broadest scope the invention can be performed inalternative ways depending on the specific needs of tumor treatment. Theabove-identified preferred embodiment describes the invention wherebythe tumor cells are either obtained from the patient to be treated orfrom tumor cell lines or tumor cell line banks. Dendritic cells arepreferably also obtained from the patient to be treated.

In a more general way, however, the present invention relates to apharmaceutical composition for inducing an immune response against tumorcells comprising

-   -   a) tumor cells which are apoptotic, whereby apoptosis is caused        by treatment with hydrostatic pressure and    -   b) dendritic cells.

It is an important aspect of the present invention that the tumor cellswhich are used in the pharmaceutical composition are apoptotic cells andnot necrotic cells. The person skilled in the art is aware of thedifferences between apoptosis versus necrosis. Cell death and subsequentpost-mortem changes, called necrosis, are integral parts of normaldevelopment and maturation cycle. Despite the importance of this processthe mechanism underlying cell death are still poorly understood althoughthere are several publications relating to the mechanisms which occurwhen a cell is dying. Apoptosis in the sense of the present invention isunderstood as a programmed, managed form of cell death whereby necrosisis an unordered and accidental form of cellular dying.

In the present invention apoptosis is understood as a mode of cell deaththat occurs under normal physiological conditions and the cell is anactive participant of its own demise. Cells undergoing apoptosis showcharacteristic morphological and biochemical features. These featuresinclude chromatine aggregation, nuclear and cytoplasmatic condensation,partitition of cytoplasm and nucleus into membrane-bound vesicles(apoptotic bodies) which contain ribosomes, morphologically intactmitochondria and nuclear material. Since these apoptotic bodies are invivo normally recognized and phagocytized by either macrophages oradjacent epithelial cells it is important that the tumor cells used inthe present method resemble as close as possible apoptotic tumor cells.Apoptosis is usually limited to individual cells and does not causeinflammatory responses.

Necrosis on the other hand occurs when cells are exposed to extremephysiological conditions which may result in damage to the plasmamembrane. Necrosis begins with an impairment of the cells' ability tomaintain hemostasis, leading to an influx of water and extracellularions. Intracellular organelles, most notably the mitochondria and theentire cell swells and ruptures. Due to the ultimate breakdown of theplasma membrane the cytoplasmic contents, including lysosomal enzymes,are released into the extracellular fluid. Therefore, in vivo necroticcell death is often associated with extensive tissue damages resultingin an intense inflammatory response. It is important that the tumorcells used in the pharmaceutical compositions are apoptotic and notnecrotic.

According to the present invention the apoptotic cells are produced by atreatment with high hydrostatic pressure. A high hydrostatic pressure asunderstood in the present invention is defined as a pressure head equalto or greater than 100 Mpa [1 MPa=10 bar=9.86923 atm=145.0377 psi]. Highhydrostatic pressure can be produced by an equipment which is forexample described in Weiss et al., Journal of Immunotoxicology, 2010, pp194-209, in particular in FIG. 1.

The high hydrostatic pressure treatment of the tumor cells is preferablyperformed in a pressure autoclave. The tumor cells are placed insuitable cryogenic vials which are filled completely with cellsuspension and closed tightly whereby the appearance of air bubbles hasto be avoided. Afterwards the vials are sealed with a flexible film(e.g. parafilm®) and the prepared vials are placed in the pressurechamber which is filled with a pressure transmitting medium. Afterwardsthe high pressure is produced by a suitable device and the cells aremaintained for a sufficient time under such high pressure.

In a preferred embodiment the hydrostatic high pressure is maintainedfor at least 10 minutes at a pressure of at least 200 MPa. In a morepreferred embodiment the tumor cells are maintained for a time range of10 minutes to 12 hours, preferably 10 minutes to 1 hour and especiallypreferred 10 to 30 minutes at a pressure in the range of 200-300 MPa,preferably 200-250 MPa.

The tumor cells to be used in the pharmaceutical composition can bederived from different sources. In one particular embodiment the tumorcells are derived from a primary tumor or from a metastatic tumor of thepatient to be treated. The tumor cells can be obtained by biopsy orsurgery. The tissue is disintegrated and the separated and purifiedtumor cells can be used immediately. It is also preferred to establish acell line of the primary tumor and to use the so obtained cells fortumor vaccination. Alternatively the tumor cells may be obtained fromsuitable tumor cell lines. Such tumor cell lines may be prepared fromthe autologous tumor. Alternatively, tumor cells may be used which arecommercially available from depository institutions such as for exampleATCC.

The other component of the pharmaceutical composition are dendriticcells. According to the present invention dendritic cells in variousstages can be used. It is possible to use either dendritic cellsdirectly obtainable from the patient by separating the dendritic cellsfrom the blood. It is, however, also possible to further classify thedendritic cells depending on their stage. In one embodiment of thepresent invention immature dendritic cells differentiated from theperipheral blood monocytes are used for the preparation of the tumorvaccine. It is known that there are three main types ofantigen-presenting cells in the peripheral lymphoid organs that canactivate T cells, namely dendritic cells, macrophages and B cells.

The most potent of these are dendritic cells whose known function is topresent foreign antigens to T cells. Immature dendritic cells arelocated in tissues throughout the body, including the skin, gut andrespiratory tract. Dendritic cells exist in two functionally andphenotypically distinct stages, immature and mature dendritic cells.Immature dendritic cells have high endocytic activity, are specializedin antigen capture and processing and reside in peripheral tissues invivo. Immature dendritic cells play a crucial role in the induction andmaintenance of peripheral tolerance. Upon exposure to pathogen-derivedproducts or innate pro-inflammatory signals, dendritic cells lose theirphagocytic activity and migrate to draining lymph nodes while becomingmature dendritic cells. Mature dendritic cells have a highantigen-presenting capability and T-cell stimulatory capacity due to theexpression of high levels of antigen-presenting, adhesion andco-stimulatory molecules as well as other dendritic cell-specificmarkers such as CD83 and DC-LAMP.

The immature dendritic cells to be used in the pharmaceuticalcomposition may be obtained from different sources. In a preferredembodiment the immature dendritic cells are differentiated from themonocytes of the patient to be treated. Alternatively, however, theimmature dendritic cells may be obtained from other sources such ascommercially available blood products obtainable from blood collectingagencies.

For the preparation of a pharmaceutical composition according to thepresent invention suitable immature dendritic cells are prepared.Dendritic cells (DCs) can be prepared by different methods and mayexhibit different properties. In a preferred embodiment of the presentinvention dendritic cells are obtained from monocytes isolated byleukapheresis.

DCs comprise less than 1% of mononuclear cells in the peripheral blood.Leukapheresis can be used to isolate approximately 10⁶ to 10⁷ dendriticcells and may be combined with positive or negative selectiontechniques. While the direct isolation of dendritic cells fromperipheral blood allows rapid preparation of dendritic cells it mayrequire repeated leukapheresis if multiple immunizations are required ina protocol.

In a preferred embodiment of the present invention monocytes areenriched from leukapheresis by adherence on plastic material. Thedendritic cells are differentiated in the presence of cytokines,preferably a cocktail of various cytokines, whereby a granulocytemacrophage-colony stimulating factor (GM-CSF) combined with interleukin4 is preferred.

A particularly preferred method of preparing dendritic cells is togenerate them ex vivo. Monocytes are dendritic cell precursors which maybe enriched from peripheral blood mononuclear cells by techniques suchas leukapheresis, plastic adherence, density gradient centrifugation,positive selection of CD14+ cells, negative selection of B- and T-cellsand combinations thereof. DC may be cultivated and differentiated bytreating an enriched precursor cell population for approximately 3-7,preferably 7 days with cytokines, in particular with granulocytemacrophage-colony stimulating factor (GM-CSF)+interleukin 4 orinterleukin 13. An advantage of this embodiment is that more than 10⁹ DCmay be prepared from a single leukapheresis product and such apreparation may be used for multiple further vaccinations bycryopreserving the DCs preparation preferably in liquid nitrogen. WhileDCs may be cultured in a variety of media it is preferred to use eitherserum-free media or media containing autologous serum. For theindustrial preparation of the pharmaceutical composition it isparticularly preferred to prepare the immature dendritic cells in alarge scale in such a manner that the occurrence of anaphylacticreactions (e.g. due to fetal calf serum) or the contamination of virusesis avoided.

In the next step of the preparation of the pharmaceutical compositionthe immature dendritic cells are loaded with the apoptotic tumor cellswhich are obtained by treatment with high hydrostatic pressure. In apreferred embodiment the immature dendritic cells which were broughtinto contact with the apoptotic tumor cells are matured by using avariety of stimuli such as the addition of Tumor Necrosis Factor α(TNF-α) or lipopolysaccharide or poly I:C.

The obtained pharmaceutical composition can be preserved for theadministration, preferably by cryopreservation. The cryopreservation ofbiospecimen is widely practiced in clinical medicine and biomedicalresearch. However, the impact of this process on cell viability andparticularly function sometimes may be underestimated. Therefore, theused method of freezing of the cell preparation prior to use in cancervaccine should be viewed with caution. The effect of cryopreservationcertainly depends on the specific cells used and it has to be examinedwhether the biological activity of the pharmaceutical composition isaltered by the cryopreservation. It may be required to add protectivecomponents like non-immunogenic polysaccharides or DMSO.

The pharmaceutical composition of the present invention can beadministered intravenously (IV), intradermally, subcutaneously orintralymphatically whereby subcutaneously is particularly preferred.

The optimal dose and frequency of immunization of the pharmaceuticalcomposition depends on the type of tumor, the age and condition of thepatient and the stage of progression of the tumor disease. In apreferred embodiment there is first applied an immunizing dose of thepharmaceutical composition which can be followed by long-termadministration of booster injections applied in intervals ranging from 2to 8 weeks.

The tumor vaccination as described herein can be applied to all forms oftumors successfully. In preferred embodiments the pharmaceuticalcompositions are for use in the treatment of cancer patients which arein a late stage of cancer, but also in the early stage of cancer. In anespecially preferred embodiment the tumor vaccination is applied topatients at a late stage of prostate cancer with hormone treatmentresistant metastatic prostate cancer. Under “early stage of cancer” suchforms of cancer are understood wherein diagnosis is possible. Frequentlythe patients do not show signs of the disease. In “late stages ofcancer” the patient suffers frequently from severe consequences of thedisease like pain or weakness.

Although it is known in the art to use adjuvant agents in tumor therapyvaccination it is preferred in the course of the present invention notto use any further adjuvant such as lipopolysaccharide, incompleteFreund's adjuvant or heat shock proteins.

It is an important aspect of the present invention that the tumor cellstreated with high hydrostatic pressure are in such a stage that theycannot grow and form a metastatic tumor after application to thepatient. This has been proven by number of experimental approaches,including the clonogenic assays.

The pharmaceutical composition as described herein can be used for thetreatment of a human by cancer immunotherapy (vaccination). The tumorcells which can be derived from a patient to be treated are brought toan apoptotic stage with the high hydrostatic pressure treatmentdescribed above. Alternatively suitable tumor cell lines are used.Immature dendritic cells are obtained preferably by leukapheresis fromthe same patient and the cells are cultured ex vivo by treatment withcytokines. A suitable amount of such immature dendritic cells (e.g.10⁷-10⁸ cells) is loaded with the apoptotic tumor cells whereby theoptimal range of immature dendritic cells:apoptotic tumor cells is 10:1to 1:1, preferably 5:1 to 3:1.

After maturation of the dendritic cells the pharmaceutical compositioncan be applied to the patient. Dendritic cells which have captured tumorcells killed by high hydrostatic pressure can be used directly for tumorvaccination. It is, however, possible to further activate or mature thecells, for example by treatment with cytokines before administration tothe patient.

According to the present invention the following materials and methodsare preferably used:

It has been shown that a treatment of ovarian and prostate cancer cellsand acute lymphoblastic leukemia cells by 10 min with high hydrostaticpressure (200 MPa) at about 21° C. leads to the induction of animmunogenic cell death of tumor cells. Tumor cells killed by HHP (highhydrostatic pressure) are immunogenic to much higher extent than tumorcells killed by anthracyclines, the only cytostatics known to induceimmunogenic cell death., or by UV-irradiation. HHP-killed immunogenictumor cells are avidly phagocytosed by antigen presenting cells andinduce their maturation even in the absence of additionalpathogen-derived stimuli, such as LPS. Antigen presenting cells loadedwith HHP killed tumor cells induce a robust CD4 and CD8 mediated tumorspecific T cell responses and do not induce potentially harmfulregulatory T cells. HHP killed tumor cells thus represent a powerfultool for clinical cancer immunotherapy approaches.

Despite the continuous introduction of new drugs and furtherimprovements of chemotherapy protocols, it is likely that, at somepoint, chemotherapy will reach its limits, and clinical efficacy willplateau. Moreover, despite the undeniable success in the treatment ofsome malignancies, in some tumors, particularly in solid tumors,chemotherapy is rarely curative. A combination of treatment modalitieshas been a standard strategy for cancer treatment, the combination ofsurgery with chemo- or radiotherapy being a classical example. Effortshould be made not only to design modern immunotherapeutic strategiesbut also to incorporate immunotherapy approaches into currentchemotherapy protocols. Chemotherapy and immunotherapy should not behenceforth considered antagonist forms of therapy, and it is conceivablethat their rational combination will substantially improve the prognosisof cancer patients.

Preferred cell lines: Acute lymphoblastic leukemia cell lines,(REH,DSMZ, Braunschweig, Germany), ovarian cancer cells (OV90, ATCC,Teddington, UK), prostate cancer cells (LNCap, ATCC, Teddington, UK)were used. All cell lines were cultured in RPMI 1640 medium (Gibco). Allmedia were supplemented with 10% heat-inactivated fetal bovine serum(Lonza), 100 U/ml penicillin and 2 mmol/L L-glutamine.

Isolation of primary tumor cells: Primary ovarian and prostate cancercells were obtained from patients undergoing surgery. Leukemic blastsfrom patients with acute lymphoblastic leukemia were obtained from thebone marrow of (ALL) patients by gradient centrifugation on Ficollgradient.

Apoptosis induction and detection: Tumor cell death was induced by 10min treatment with high hydrostatic pressure. For comparative teststumor cell death was induced by UV light exposure. In this case anenergy of 7.6 J/cm² was applied for 10 min. Cell death was assessed byannexin V fluorescein isothiocyanate staining. Briefly, 2×10⁵ cells persample were collected, washed in PBS, pelleted, and resuspended in anincubation buffer containing annexin V fluorescein isothiocyanateantibody. The samples were kept in the dark and incubated for 15 minbefore the addition of another 400 μl of 0.1% propidium iodideincubation buffer and subsequent analysis on an Ariafluorescence-activated cell sorter (BD Bioscience) using FlowJosoftware.

Flow cytometric analysis of hsp70, hsp90 and CRT (calreticulin) on thecell surface: A total of 10⁵ cells were plated in 12-well plates andtreated the following day with the indicated agents or were—as acontrol—UV-irradiated (7.6 J/cm²) for 6, 12 or 24 h or were treated for10 min with high hydrostatic pressure at 21 degrees centigrade's. Thecells were collected and washed twice with PBS. The cells were incubatedfor 30 min with primary antibody diluted in cold blocking buffer (2%fetal bovine serum in PBS), followed by washing and incubation with theAlexa 648-conjugated monoclonal secondary antibody in a blockingsolution. Each sample was then analyzed by FACScan (BD Bioscience) toidentify cell surface hsp70, hsp90 and CRT.

Detection of HMGB1 release: HMGB1 enzyme-linked immunosorbent assay IIkits were obtained from SHINO-TEST CORPORATION (Tokyo, Japan). REHcells, OV90 cells, LNCap cells, primary ovarian cells and leukemicblasts (10⁶) were plated in 1 ml full medium appropriate for the celltype. Supernatants were collected at different time points, dying tumorcells were removed by centrifugation, and the supernatants were isolatedand frozen immediately. Quantification of HMGB1 in the supernatants wasassessed by enzyme-linked immunosorbent assay according to themanufacturer's instructions.

Fluorescent microscopy: Immunofluorescence: For surface detection ofCRT, the cells were placed on ice, washed twice with PBS and fixed in0.25% paraformaldehyde in PBS for 5 min. The cells were then washedtwice in PBS, and a primary antibody diluted in cold blocking buffer wasadded for 30 min. After two washes in cold PBS, the cells were incubatedfor 30 min with the appropriate Alexa 648-conjugated secondary antibody.The cells were fixed with 4% paraformaldehyde for 20 min, washed in PBSfor 20 min and mounted on slides.

For phagocytosis, the DCs were stained with Vybrant® DiO cell labelingsolution (Invitrogen). The tumor cells were stained with Vybrant® DiIcell labeling solution (Invitrogen) and cultured in the presence ofanthracyclins, UV light exposure or 10 min treatment with highhydrostatic pressure at 21 degrees centigrade's . Immature DCs (day 5)were fed tumor cells at a DC/tumor cell ratio of 1:5. The cells werefixed with 4% paraformaldehyde for 20 min, washed in PBS for 20 min andmounted on slides with ProLong Gold antifade reagent (Invitrogen).

Generation of tumor-loaded DCs and induction of tumor cell death: DCswere generated by culture of purified CD14⁺ cells isolated from buffycoats in the presence of granulocyte-macrophage colony-stimulatingfactor (GM-CSF) (Gentaur, Brussels, Belgium) and interleukin-4 (IL-4)(Gentaur, Brussels, Belgium). Tumor cells were killed by 10 min.treatment with high hydrostatic pressure at 21 degrees centigrade's,or—as controls—by UV irradiation or by anthracyclines. The extent ofapoptosis was monitored by annexin V/PI staining. The cells wereextensively washed prior to feeding to DCs. Immature DCs (day 5) werefed tumor cells at a DC/tumor cell ratio of 1:5. In some experiments,pulsed DCs were stimulated with 100 ng/ml of lipopolysaccharide (LPS)(Sigma) for 12 h or 25 μg/ml of Poly I:C (obtained from Invivogen).

FACS analysis of DC phenotype after interaction with killed tumor cells:The phenotype of DCs cultured with tumor cells was monitored by flowcytometry. Tumor cells were killed by a selected cytostatic agent or UVirradiation (comparative examples) or 10 min treatment with highhydrostatic pressure at 21 degrees centigrades (according to the presentinvention) and were cocultured for 24 h with immature DCs. For someexperiments, the DCs and tumor cells were dye-labeled before cocultureto monitor phagocytosis. Monoclonal antibodies (mAbs) against thefollowing molecules were used: CD80-FITC, CD83-FITC, CD86-PE, CD14-PE(Immunotech, Marseille, France), CD11c-PE, HLA-DR (BD Biosciences, SanJose, Calif.).

The DCs were stained for 30 minutes at 4° C., washed twice inphosphate-buffered saline (PBS) and analyzed using FACS Aria (BDBiosciences) using FlowJo software. The DCs were gated according to theFSC and SSC properties. The appropriate isotype controls were included,and 50000 viable DCs were acquired for each experiment.

Evaluation of IFN-γ producing tumor-specific T cells; Unpulsed or tumorcells-loaded DCs were added to autologous T cells at a ratio of 1:10 ondays 0 and 7 of culture. IL-2 (25-50 international units/mL; PeproTech)was added on days 2 and 7 of culture. The cultures were tested for thepresence of tumor-specific T cells 7 to 9 days after the laststimulation with DCs. The induction of tumor-reactive, interferon(IFN)-γ-producing T cells of prostate specific antigen (PSA) reactive Tcells by tumor-loaded DCs was determined by flow cytometry. The T cellswere stained with anti-human CD8/IFN-γ. Frequency of regulatory Tlymphocytes in the culture was analyzed by staining with CD4/CD25 andFoxP3. Regulatory T cells were identified by flow cytometry as CD4positive, CD25 positive and FoxP3 positive.

The invention and the results obtained by the experiments areillustrated by the Figures;

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

The schematic drawing shows how a pharmaceutical composition of thepresent invention can be obtained. Tumor cells obtained either from thepatient or from cell lines are treated with high pressure whereby thecells become apoptotic.

Dendritic cells are isolated via leukapheresis, Immature dendritic cellsand apoptotic tumor cells are combined whereby mature dendritic cellsare produced which can be used as vaccine,

FIG. 2

High hydrostatic pressure induces the expression of heat shock proteinson human tumor cells. The summary of a total of 5 experiments isshown. * P value for comparison with irradiated tumor cells, P<0,05. Thetime dependent expression of the markers HSP70, HSP90 and calreticulinon two tumor cell lines (OV90 and LNCap) caused by different treatmentsis shown.

FIG. 3

High hydrostatic pressure induces the release of HMGB1 (high-mobilitygroup protein B1) from treated tumor cells (OV90 and LNCap). HMGB1 is acytokine mediator of inflammation. The summary of a total of 5experiments is shown. *P value for comparison with irradiated tumorcells, P<0.05. FIG. 3 shows that concerning the time dependent releaseof HMBG1 the HHP treatment is much more effective than otherconventional treatments.

FIG. 4

The kinetics of phagocytosis of high hydrostatic pressure treated tumorcells by immature DCs. Summary of 5 independent experiments andrepresentative results are shown. In the experiment either OV90 or LNCaptumor cells were used. HHP treatment is compared with UV treatment at 0°C. and 37° C.

FIG. 5

The phenotype of dendritic cells based on the markers OD86 and HLA-DRafter interaction with high hydrostatic pressure-killed tumor cells(OV90 and LNCap) is shown. Day 5 immature DCs were cultured for 24 hwith tumor cells killed by HHP or irradiation. After 24 h, theexpression of maturation associated molecules on DCs was analyzed byflow cytometry. LPS was used as control. The mean fluorescence intensity(MFI)are shown. *P value for comparison with irradiated tumorcell-loaded DCs, P<0.05.

FIG. 6

The induction of tumor-specific T cells by dendritic cells loaded withhydrostatic pressure killed tumor cells (LNCap and OV90) is comparedwith dendritic cells loaded with tumor cells killed by UV irradiation.The data show a summary of five independent experiments. *P value forcomparison with irradiated tumor cells, P<0,05.

FIG. 7

FIG. 7 demonstrates the superiority of the treatment of tumor cells withhigh hydrostatic pressure (HHP) compared with tumor cells killed by UVirradiation (UV irr). The tests have been performed with prostate cancercell line (LNCap) and with ovarian cancer cell line (OV90). Controlshave been performed with dendritic cells alone and cells stimulated withPoly I:C.

The results summarized in FIG. 7 show the induction of prostate specificantigen (PSA)-specific T cells by dendritic cells loaded with highhydrostatic pressure killed tumor cells (LNCap and OV90, respectively).A comparison was made between high hydrostatic pressure killed tumorcells alone and dendritic cells loaded with tumor cells killed by UVirradiation. The data presented in FIG. 7 show a summary of fiveindependent experiments. *P value for comparison with irradiated tumorcells, P<0.05. FIG. 7 summarizes the results obtained in example 7.

FIG. 8

The induction of regulatory T cells by high hydrostatic pressure killedtumor cells is compared with the induction of Tregs by UV irradiatedtumor cells. The data show a summary of five independent experiments.

The experiments summarized in FIG. 8 show that the teaching of thepresent invention can be applied to different types of tumors. The upperpart of FIG. 8 shows the experiments performed with ovarian cancer cells(OV90). The lower part shows the experiments performed with prostatecancer cell line (LNCap). In the experiments the concentration of Fox P3(Forkhead Box P3) has been determined in order to further differentiatethe regulatory T cells (Tregs). The experiments show that tumor cellstreated according to the invention with HHP do induce lower numbers ofregulatory T cells than UV irradiated tumor cells.

FIG. 9

The results of an in vivo study are shown wherein patients were treatedwith a tumor vaccination as disclosed herein. All patients had radicalprostatectomy or radiotherapy. As relevant parameter the PSA doublingtime has been determined. According to Antonarakis et al., BJU Int.2011, 108(3), p. 378-385, the PSA doubling time is the strongestdeterminant of metastatic free survival time and overall survival timeof patients with prostate specific antigen (PSA)-recurrent prostatecancer. PSA doubling time means the time difference wherein the PSAvalue is doubled. The higher the PSA doubling time is, the better thesurvival prospect for the treated patient is. By applying the tumorvaccination of the present invention the PSA doubling time could besubstantially prolonged.

*P value for comparison with irradiated tumor cells, P<0,05.

FIG. 10

FIG. 10 is a Kaplan-Meier survival curve of patients at a late stage ofprostate cancer which were treated according to the present invention.

In the Kaplan-Meier survival curve each death of a patient causes a dropof the percent survival starting from 100% to lower values. The Halabinomogram is the normally expected reduction of survivors whereby themedium survival time is 12 months.

The active cancer immunotherapy using the cancer vaccine as describedherein results in a prolongation of the medium survival time to 23months.

The present invention is further illustrated by the following exampleswhich are, however, not limiting:

EXAMPLES Example 1

Expression of Immunogenic Cell Death Markers hsp70, hsp90 andCalreticulin by Human Cancer Cell Lines and Human Primary Tumor CellsAfter the Treatment with High Hydrostatic Pressure

Leukemic, ovarian and prostate cancer cell lines and primary tumor cellswere treated for 10 min with high hydrostatic pressure (HHP, 200 MPa) at21 degrees centigrade's and the expression of the known immunogenic celldeath markers hsp70, hsp90 and calreticulin was monitored at 6, 12 and24 h. Significant expression of calreticulin, hsp70 and hsp90 wasdetected 6, 12 and 24 h after HHP treatment for all tested tumor models.The expression of immunogenic molecules was significantly higher thanthe expression induced by anthracyclins, the only known inducers ofimmunogenic cell death (FIG. 2). Increased expression of calreticulinand heat shock proteins after HHP treatment was accompanied by theirtranslocation to the cell surface. HHP treatment also induced a rapidand substantial release of HMGB1, a soluble marker of immunogenic celldeath. Release of HMGB1 was much higher than in the case of UVirradiation or anthracyclines. (FIG. 3).

Maximal release of HMGB1 nuclear protein was detected 48 h after theinduction of tumor cell death.

Example 2

Treatment of Tumor Cells by High Hydrostatic Pressure Increases TheirPhagocytosis by Antigen Presenting Cells

In view of the established role of calreticulin as an ‘eat me’ signal,the rate of phagocytosis of tumor cells killed by high hydrostaticpressure by dendritic cells (DCs) was investigated, the most efficientantigen presenting cells that are crucial for the initiation of animmune response. High hydrostatic pressure treated tumor cells werephagocytosed at faster rate and to a higher extent than the tumor cellskilled by other modalities, such as anthracyclines or UV irradiation.After 12 h, the extent of phagocytosis of leukemic cells treated withHHP was 4-fold higher than of cells killed by UV irradiation (FIGS. 4aand 4b ).

Example 3

Phagocytosis of High Hydrostatic Pressure-Treated Tumor Cells Inducesthe Maturation of DCs

The ability of DCs to activate the immune response depends on theiractivation status and the expression of costimulatory molecules. Innormal circumstances the most efficient maturation of DCs is induced bymolecules derived from pathogens, such as lipopolysacharide (LPS) fromGram negative bacteria. Only activated (mature) DCs that express highlevels of costimulatory molecules can initiate the immune response. Weanalyzed the phenotype of DCs that phagocytosed tumor cells killed bythe HHP. The interaction of DCs with HHP-treated tumor cells induced theupregulation of costimulatory molecules (CD86, CD83) and maturationassociated molecules (HLA-DR) to a similar extent as activation by LPS(FIG. 5). Thus tumor cells killed by HHP can induce DCs maturationcomparable to pathogen derived LPS.

Example 4

DCs Presenting High Hydrostatic Pressure Treated Tumor Cells InduceTumor-Specific T Cells and Induce Low Numbers of Inhibitory Regulatory TCells

To investigate whether tumor cells treated with HHP and expressingimmunogenic cell death markers induce anti-tumor immunity, we evaluatedthe ability of tumor cell-loaded DCs to activate tumor cell-specific Tcell responses. Tumor cells killed by HHP were cocultured with immatureDCs with or without subsequent maturation with LPS. These DCs were thenused as stimulators of autologous T cells, and the frequency ofIFN-γ-producing T cells was analyzed one week later after restimulationwith tumor cell-loaded DCs. DCs pulsed with HHP killed tumor cellsinduced a greater number of tumor-specific IFN-γ-producing T cells incomparison with DCs pulsed with irradiated cells, even in the absence ofadditional maturation stimulus (LPS).

Additionally, the frequency of regulatory T cells (Tregs) induced in DCand T cell cocultures was also tested. Induction of Tregs is undesirablein the case of tumor immunotherapy as Tregs inhibit the immune responsedirected against the tumor. DCs pulsed with tumor cells killed by HHPhad a lower capacity to expand regulatory T cells when compared withboth immature DCs and LPS-activated DCs (FIG. 8). The FoxP3 surfacemarker is specific for regulatory T cells.

Example 5

Active cellular immunotherapy can be administered as a single treatmentmodality in the case of minimal residual disease after primary treatmentof the tumor by surgery or radiotherapy. In prostate cancer it mayconcern patients with signs of biochemical relapse (increasing levels ofprostate-specific-antigen PSA in the peripheral blood measured byultrasensitive method),

The best results of the present invention can be obtained when theprimary tumor is removed from the patient by surgery. The pharmaceuticalcomposition as described in the present application can be produced fromthe tumor cells which have been isolated from the tumor tissue or fromtumor cell lines.

A patient (68 years old) suffering from prostate cancer was diagnosed atan early stage of the tumor development. Tumor was removed but fewmonths after the surgery rising levels of PSA were detected. The patientthus underwent leukapheresis and immature dendritic cells weredifferentiated from isolated monocytes. Tumor cells from the prostatecancer cell line were rendered apoptotic treatment with high hydrostaticpressure as described herein and the apoptotic tumor cells were broughtinto contact with the immature dendritic cells in order to prepare thevaccine composition.

The pharmaceutical composition was divided into aliquots that werefrozen in the liquid nitrogen until use. The first application of thetumor vaccination occurred 4 weeks after the detection of thebiochemical relapse of the prostate cancer. Booster applicationsfollowed every four weeks for a period of one year.

Vaccination induced an immune response against the small number ofsurviving tumor cells that has lead to a substantial slowing down ofregrowth of tumor cells and resulted in the prolongation of the survivalof the patient.

Example 6

In advanced cancer patients, active cellular immunotherapy should becombined with chemotherapy (i.e. docetaxel in prostate cancer) accordingto the concept of chemo-immunotherapy.

A patient (76 years old) suffering from advanced prostate cancer wastreated according to the present invention. The usual chemotherapy wascombined with the active cellular immunotherapy as disclosed herein. Thepatient has been treated at the age of 65 years with prostate tumor.After removal of the tumor by surgery and hormone treatment the level ofPSA (prostate specific antigen) was kept at a low level showing that theprostate cancer cells did not grow. After 12 months of hormone therapymetastatic prostate cancer developed at several positions in the body(in particular in the bones) and the tumor became hormone refractory.The patient was approved for the treatment of hormone refractoryprostate cancer with docetaxel in combination with active cellularimmunotherapy based on dendritic cells.

Before the chemotherapy started, immature dendritic cells were generatedfrom monocytes obtained during leukapheresis. Tumor cells from prostatecancer cell lines were treated with hydrostatic pressure for 30 minutesat a pressure of 210 MPa at 21° C. 10⁹ tumor cells treated according tothe present invention were used to pulse 10⁹ immature dendritic cellsand aliquots of the mature dendritic cells which have been pulsed beforewith those tumor cells were deep-frozen in liquid nitrogen and used forlater applications.

Active cancer immunotherapy was administered every 4-6 weeks inalternate cycles with standard chemotherapy by docetaxel and alone(after the end of docetaxel treatment) for a period of one year.Combined chemoimmunotherapy led to the stabilization of the disease,decrease in the intensity of bone marrow metastases and longer thanexpected survival. Patient currently survives for over three years,compared to the expected survival of 6 months at the beginning of thetherapy.

Example 7

In Vitro Experiment Showing the Superiority of HHP Killed Tumor CellsVersus UV Killed Tumor Cells

In the in vitro experiments the ability of immature dendritic cells,poly I:C activated mature dendritic cells, and dendritic cells loadedwith tumor cells which were either HHP treated or UV irradiated waschecked with regard to their ability of induce tumor specific immunity.Tumor specific immunity was measured as percent tumor specific T celllymphocytes.

Dendritic cells with HHP killed tumor cells were directly compared withHHP killed tumor cells alone and dendritic cells loaded with tumor cellskilled by UV irradiation. The results of the experiments are shown inFIG. 7.

In order to test the capacity to induce tumor-specific T cells unpulsedor loaded with tumor cells dendritic cells were added to autologous Tcells at a ratio of 1:10 on days 0 and 7 of culture. 25-50 internationalunits/mL of IL2 (PeproTech) were added on days 2 and 7 to the culture.The cultures were tested for the presence of tumor specific T cells 7-9days after the last stimulation with DCs. The induction oftumor-reactive, interferon (IFN)-γ-producing T cells of prostatespecific antigen (PSA) reactive T cells by tumor-loaded DCs wasdetermined by flow cytometry. The T cells were stained with anti-humanCD8/IFN-γ.

The induction of prostate specific antigen (PSA)-specific T cells bydendritic cells loaded with high hydrostatic pressure killed tumor cells(LNCap) is compared with high hydrostatic pressure killed tumor cellsalone and with dendritic cells loaded with tumor cells killed by UVirradiation.

The results of the experiments are shown in FIG. 7. The upper part ofFIG. 7 shows that DCs loaded with HHP killed tumor cells can inducetumor specific T cells even in the absence of a maturation signal. DCsloaded with tumor cells killed by UV treatment or HHP killed tumor cellsalone do not induce tumor immunity. It is surprising that only HHPtreated tumor cells (according to the invention) and immature dendriticcells can induce tumor specific immune response whereas this resultcannot be obtained by UV treated tumor cells and immature dendriticcells. Without wishing to be bound to a theory it seems that only theHHP treated tumor cells can together with immature dendritic cellsinduce the tumor specific T cell immune response. The HHP treated tumorcells seem to act as a kind of activator of the immature dendritic cellswhereas UV treated tumor cells do not have this effect.

The lower part of FIG. 7 shows that when Poly I:C treatment is appliedthe treated HHP tumor cells can better induce specific T celllymphocytes than tumor cells irradiated with UV.

Example 8

In Vivo Data Obtained with the Tumor Vaccination According to thePresent Invention

Dendritic cells were obtained from a cohort of patients similar to thoseas described above. The dendritic cells were pulsed with killed tumorcells as described above and the tumor vaccination was administeredrepeatedly in up to 12 doses in 4-6 weeks intervals to patients with abiochemical relapse of the prostate cancer after radical prostatectomyor radiotherapy. The progression of the disease in each single patienthas been evaluated by the PSA doubling time. Under PSA doubling time thetime period is understood which is required for the PSA value to double.PSA doubling time has been shown as the strongest and most reliabledeterminant of the overall survival and metastatic free survival in menwith prostate cancer. Short PSA doubling time correlates with ashortened survival and with shortened time to metastasis appearance(Antonarakis et al., BJU Int., 2012, 108(3); pp 378-385.

As shown in FIG. 9 the continuous administration of the tumorvaccination according to the present invention in patients withbiochemical relapse of the prostate cancer after radical prostatectomyor radiotherapy leads to a significant prolongation of the PSA doublingtime. It has been found that by using the tumor vaccination as disclosedherein mean PSA doubling time increases from 5 months before theinitiation of cancer immunotherapy to 30 months after 12 months ofimmunotherapy. This represents a significant benefit to patients withthe biochemical relapse of the prostate cancer.

Example 9

Clinical Trial with Patients in Late Stage of Prostate Cancer

In this clinical trial dendritic cells were pulsed with killed tumorcells as described herein. The tumor vaccination was administeredrepeatedly to patients at a later stage of the prostate cancer. Saidpatients suffered from castration resistant metastatic prostate cancer.In those patients cancer immunotherapy was administered in alternatedosing schedule with docetaxel chemotherapy.

The survival of the treated cohort was compared to the historical cohortor to the survival estimated by Halabi nomogram. It has been shown thatthe continuous administration of active cancer immunotherapysignificantly prolongs the survival time of treated patients (mediansurvival of 23 months) compared with the cohort of the historicalcontrols based on the expected survival calculated by Halabi nomogram(13 months).

This experiment proves that the tumor vaccination of the presentinvention substantially extends the survival time of patients which arein a late state of prostate cancer. The average survival expectation ofsuch patients is 13 months without treatment compared to 23 months aftertreatment with tumor vaccination according to the present invention.This represents a substantial improvement for such patients which areextremely difficult to medicate successfully.

1-28. (canceled)
 29. A method of treating a human patient by cancervaccination, wherein said method comprises the step of administering apharmaceutical composition for inducing an immune response against tumorcells in a patient in need thereof comprising: a) tumor cells isolatedfrom tumor cell lines or from a tumor tissue obtained from the patientto be treated and rendered apoptotic from in vitro treatment for 10minutes to 2 hours with a high hydrostatic pressure (HHP) that rangesfrom 100 MPa to 300 MPa and b) human dendritic cells having saidapoptotic tumor cells loaded thereon ex vivo, wherein said apoptotictumor cells induce a higher level of phagocytosis by said dendriticcells as compared to tumor cells treated with UV irradiation, furtherwherein prior to loading, said apoptotic tumor cells are confirmed byclonogenic assay to be incapable of growing and forming a metastatictumor after application to the patient, and further wherein theresulting dendritic cells have an at least 1.3 fold increase ofactivation markers CD86 and/or HLA-DR compared to dendritic cells loadedwith tumor cells treated with UV irradiation, to the patient as a cancervaccine.
 30. The method according to claim 29, wherein the tumor cellsare treated with high hydrostatic pressure (HHP) ranging from 200 MPa to300 MPa.
 31. The composition according to claim 29, wherein the tumorcells are treated with high hydrostatic pressure (HHP) applied to saidtumor cells for 10 minutes to 1 hour.
 32. The method according to claim29, wherein the apoptotic tumor cells are not necrotic.
 33. The methodaccording to claim 29, wherein the tumor cells are derived from tumorcell lines.
 34. The method according to claim 29, wherein the tumorcells are derived from a tumor isolated from the patient to be treated.35. The method according to claim 29, wherein the dendritic cells areimmature dendritic cells.
 36. A method of treating a human patient bycancer vaccination, wherein said method comprises the step ofadministering an ex vivo generated composition for inducing an immuneresponse against tumor cells in a patient in need thereof, saidcomposition comprising mature human dendritic cells produced by (a)pulsing ex vivo immature dendritic cells with tumor cells that, prior topulsing, are first obtained from a tumor cell line or from a tumortissue isolated from the patient being treated and then renderedapoptotic from in vitro treatment for 10 to 2 hours with a highhydrostatic pressure (HHP) that ranges from 100 MPa to 300 MPa, andwherein the resulting HHP treated tumor cells are enriched forexpression of HSP70, HSP90 or calreticulin compared to expression intumor cells treated by UV irradiation or anthracyclines and (b) allowingsaid ex vivo dendritic cells to mature, wherein said mature dendriticcells are able to induce CD4 and CD8-mediated tumor specific T-cellresponses without inducing potentially harmful regulatory T-cells atlevels that could inhibit the anti-tumor immune response, to the patientas a cancer vaccine.
 37. The method according to claim 36, wherein saidmature dendritic cells express elevated levels of co-stimulatory andmaturation-associated molecules as compared to dendritic cells culturedwith tumor cells killed by UV irradiation.
 38. The method according toclaim 37, wherein said co-stimulatory and maturation-associatedmolecules are CD86 and/or HLA-DR.
 39. The method according to claim 36,wherein prior to pulsing, said apoptotic tumor cells are confirmed byclonogenic assay to be incapable of growing and forming a metastatictumor after application to the patient.
 40. The method according toclaim 36, wherein said mature dendritic cells induce lower numbers ofregulatory T-cells and higher numbers of tumor antigen-specific T-cellsas compared to dendritic cells pulsed with tumor cells treated with UVirradiation.
 41. The method according to claim 36, wherein the immaturedendritic cell to tumor cell ratio ranges from 1:1 to 10:1.
 42. Themethod according to claim 36, wherein the immature dendritic cell totumor cell ratio ranges from 4:1 to 6:1.
 43. The method according toclaim 36, wherein said composition is formulated for intravenous (IV),intradermal, subcutaneous or intralymphatic administration to saidpatient.
 44. The method according to claim 36, wherein said maturedendritic cells are capable of maintaining a low level of Forkhead BoxP3 (FoxP3) positive T regulatory cells of 2% or less.
 45. The methodaccording to claim 36, wherein said HHP treated tumor cells exhibit atleast a difference of 1.3 in fold changes of calreticulin expression,compared to calreticulin expression in tumor cells treated by UVirradiation or anthracyclines.
 46. The method according to claim 29,wherein the loaded dendritic cells are capable of maintaining a lowlevel of Forkhead Box P3 (FoxP3) positive T regulatory cells of 2% orless.
 47. The method according to claim 36, wherein the tumor cells aretreated with high hydrostatic pressure (HHP) ranging from 200 MPa to 300MPa.
 48. The composition according to claim 36, wherein the tumor cellsare treated with high hydrostatic pressure (HHP) applied to said tumorcells for 10 minutes to 1 hour.